Hybridoma [1] and phage-display recombinant antibody systems [2] are currently the predominant methods for isolating monoclonal Abs. Display of recombinant antibodies (Ab) on the surface of bacteriophage M13 has numerous advantages compared to conventional hybridoma technology. When combined with the use of large non-immune libraries, phage Ab selection represents a rich source of binders that can be isolated in a fraction of the time needed for hybridoma-based approaches. As an in vitro selection methods, it permits the selection of binders against toxic or highly conserved antigens, which is not easily performed using the conventional hybridoma techniques and importantly, it can be used to isolate fully human antibody fragments [3]. Consequently, phage display rapidly became an established procedure for the isolation of binders against a wide variety of antigens.
Phage display-based antibody isolation typically relies on the use of recombinant proteins for several steps, including immunizations (if needed), library enrichment by selection on immobilized antigen, screening, and characterization of antibodies specificity and affinity [4]. This procedure is efficient but depends on the availability of purified recombinant proteins. Unfortunately, some surface molecules, such as G-protein coupled receptors, cannot be easily expressed and purified in a native conformation. Some molecules with large extracellular domains may adopt a specific conformation due to interaction with other cell surface proteins, thereby forming complexes that are cumbersome to produce by recombinant expression. Moreover, many standard screening practices, such as the adsorption of recombinant proteins on plastic, may significantly alter protein conformations [5]. For these reasons, Abs selected on the basis of binding to a recombinant protein may not bind the native conformation of this protein. It is thus of high interest to develop procedures entirely based on the use of intact cells expressing the receptor of choice. However, in this case, an extra step is necessary to enrich for phage-Abs binding to the receptor of interest rather than to other cell surface proteins. Since selection steps are performed in vitro, it is possible to influence the outcome of a selection by performing some additional steps such as deletion steps (also named negative selection) prior to positive selections to remove unwanted specificities or cross-reactions [6], or competitive elution using a ligand or an antibody to favor the selection of binders against a precise epitope [7].
Along this line, it would be of very high interest to establish a procedure able to reliably guide the selection toward an unknown but relevant antigen within a complex mixture, such as a tumor maker overexpressed at the surface of intact cells, or in a cell lysate. Indeed, during the past two decades, there has been a growing interest in approaches aiming at discovering new diagnosis biomarkers and identifying new potential surface markers for targeted therapy. Several studies have described the use of phage display and libraries of recombinant antibodies for the isolation of tumor specific binders [8-14], leading in some cases to the identification of new tumor markers [15, 16]. Most of these strategies are based on the use of depletion steps on normal samples followed by a selection step on the tumor sample. Unfortunately, this procedure often leads to inconsistent results and its efficiency can be a limiting factor in complex situation such as the selection of antibodies against unknown overexpressed tumor antigens.